Supporting Information Table S1. Bacterial Strains and Plasmids used in this study
Strains or Plasmids
Genotype or Description
Source or Reference
E. coli strains
E. coli cloni10G
F- mcrA D(mrr-hsdRMS-mcrBC) f80dlacZÄM15 D lacX74 Lucigen
endA1 recA1araD139 D (ara, leu)7697 galU galK rpsL nupG
- tonA ; E. coli derivative ultra competent cells used for
general cloning.
E. coli BL21(DE3)Star
F– ompT hsdSB(rB–, mB–)gal dcm rne131(DE3); used to Stratagene
express recombinant proteins
E. coli BTH101
F–, cya-99, araD139, galE15, galK16, rpsL1 (Str r), hsdR2,
[1]
mcrA1, mcrB1 used to perform BTH assay.
Mycobacterial strains
M. smegmatis mc2155
ept-1
[2]
M. smegmatis
mc2155ΔparB
mc2155 strain deleted from parB
[3]
E. coli plasmids
pUC57
pUC19 derivative,
ExoIII deletions
designed for cloning and generation of Genscript
pUC57_parBAla
pUC57 derivative used by Genscript to deliver synthesized This study
parB10Ala gene where phosphorylated residues were substituted
by Alanine (S5A/T32A/T41A/T53A/T110A/T195A/S239A/
T254A/T300A/ T303A)
pUC57_ parBAsp
pUC57 derivative used by Genscript to deliver synthesized This study
parB10Asp gene where phosphorylated residues were substituted
by Aspartate (S5D/T32D/T41D/T53D/T110D/T195D/S239D/
T254D /T300D/ T303D)
pETPhos
pET-15b (Novagen) derivative including the replacement of [4]
the thrombin site coding sequence with a tobacco etch virus
(TEV) protease site and Ser to Gly mutagenesis in the N-term
His-tag
pETPhos_parB
pETPhos derivative used to express His-tagged fusion of This study
Mycobacterium tuberculosis ParB WT in E. coli
pETPhos_parBAla
pETPhos derivative used to express His-tagged fusion of This study
Mycobacterium tuberculosis ParB10Ala in E. coli
pETPhos_parBAsp
pETPhos derivative used to express His-tagged fusion of This study
Mycobacterium tuberculosis ParB10Asp in E. coli
pCDFDuet-1
pET vector derivative designed for the co-expression of two Novagen
proteins under T7lac promoter induction
1
pDuet_parB/pknB
pET vector derivative used for the co-expression of PknB and This study
His ParB proteins under T7lac promoter induction
pDuet_parB/pknF
pET vector derivative used for the co-expression of PknF and This study
His ParB proteins under T7lac promoter induction
pDuet_parB/pknH
pET vector derivative used for the co-expression of PknH and This study
His ParB proteins under T7lac promoter induction
pKT25
pSU40 derivative, allows expression of N-terminal T25 fragment [1]
(amino acids 1 to 224 of CyaA) fusion protein under lac
promoter in E. coli.
pKNT25
pSU40 derivative, allows expression of C-terminal T25 fragment [1]
(amino acids 1 to 224 of CyaA) fusion protein under lac
promoter in E. coli.
pUT18
pUC19 derivative, allows expression of N-terminal T18 [1]
fragment (amino acids 225 to 399 of CyaA) fusion protein under
lac promoter in E. coli.
pUT18c
pUC19 derivative, allows expression of C-terminal T18 [1]
fragment (amino acids 225 to 399 of CyaA) fusion protein under
lac promoter in E. coli.
pKT25_parBWT
pKT25 derivative used to express N-terminal CyaA T25 fusion This study
of M. tuberculosis ParB WT in E. coli
pKT25_parBAla
pKT25 derivative used to express N-terminal CyaA T25 fusion This study
of M. tuberculosis ParB Ala in E. coli
pKT25_parBAsp
pKT25 derivative used to express N-terminal CyaA T25 fusion This study
of M.tuberculosis ParB Asp in E. coli
pKT25_parA
pKT25 derivative used to express N-terminal CyaA T25 fusion This study
of M. tuberculosis ParA in E. coli
pNKT25_parBWT
pNKT25 derivative used to express C-terminal
fusion of M. tuberculosis ParB WT in E. coli
CyaA T25 This study
pNKT25_parBAla
pNKT25 derivative used to express C-terminal
fusion of M. tuberculosis ParB Ala in E. coli
CyaA T25 This study
pNKT25_parBAsp
pNKT25 derivative used to express C-terminal
fusion of Mtuberculosis ParB Asp in E. coli
CyaA T25 This study
pNKT25_parA
pNKT25 derivative used to express C-terminal T25 of CyaA This study
fusion of M tuberculosis ParA in E. coli
pUT18_parBWT
pUT18 derivative used to express N-terminal CyaA T18 fusion This study
of M tuberculosis ParB WT fused to in E. coli
2
pUT18_parBAla
pUT18 derivative used to express N-terminal CyaA T18 fusion This study
of M tuberculosis ParB Ala in E. coli
pUT18_parBAsp
pUT18 derivative used to express N-terminal CyaA T18 fusion This study
of M tuberculosis ParB Asp in E. coli
pUT18_parA
pUT18 derivative used to express N-terminal CyaA T18 fusion This study
of M tuberculosis ParA in E. coli
pUT18c_parBWT
pUT18c derivative used to express C-terminal CyaA T18 fusion This study
of M tuberculosis ParB WT fused to in E. coli
pUT18c_parBAla
pUT18c derivative used to express C-terminal CyaA T18 fusion This study
of M tuberculosis ParB Ala in E. coli
pUT18c_parBAsp
pUT18c derivative used to express C-terminal CyaA T18 fusion This study
of M tuberculosis ParB Asp in E. coli
pUT18c_parA
pUT18c derivative used to express C-terminal CyaA T18 fusion This study
of M tuberculosis ParA in E. coli
Mycobacterial shuttle
plasmids
pVV16
E. coli/mycobacterial shuttle vector, allows expression of C- [5]
terminal His-tagged proteins, derived from pMV261 and
containing the hsp60 constitutive promoter
pVV16_parB-egfp
pVV16 derivative used to express C-terminal EGFP-tagged This Study
fusion of ParB in mycobacteria
pVV16_parBAla-egfp
pVV16 derivative used to express EGFP-tagged fusion of ParB This Study
phosphorylation sites mutated in alanine residues
(S5A/T32A/T41A/T53A/T110A/T195A/S239A/T254A/T300A/
T303A) in mycobacteria
pVV16_parBAsp-egfp
pVV16 derivative used to express EGFP-tagged fusion of ParB This Study
phosphorylation sites mutated in aspartate residues
(S5D/T32D/T41D/T53D/T110D/T195D/S239D/T254D/T300D/
T303D) in mycobacteria
3